CN111821434A - Use of anti-PD-1 antibody in the preparation of medicaments for the treatment of solid tumors - Google Patents

Abstract

The present invention relates to the use of anti-PD-1 antibodies in the treatment of tumors. The invention also relates to the use of a reagent for detecting gene amplification in the chromosome 11q13 region in a test kit for predicting the effect of anti-PD-1 antibody and/or antigen-binding fragment therapy in a patient with a tumor.

Description

Use of anti-PD-1 antibody in the preparation of medicaments for the treatment of solid tumors

technical field

The present invention relates to the use of anti-PD-1 antibodies in the treatment of tumors. In particular, the present invention relates to the use of anti-PD-1 antibodies in the treatment of solid tumors, especially esophageal cancer, more preferably esophageal squamous cell carcinoma (ESCC); and the use of anti-PD-1 antibodies in the preparation of therapeutic entities Tumors, especially esophageal cancer, acral cutaneous melanoma, more preferably esophageal squamous cell carcinoma (ESCC) use in medicine; and use of biomarkers to predict the use of anti-PD-1 antibodies in the treatment of solid tumors, Especially the use in esophageal cancer, more preferably the method of therapeutic effect in esophageal squamous cell carcinoma (ESCC); and the use of the combination therapy or composition comprising the anti-PD-1 antibody in the treatment of solid tumors.

Background technique

Immune escape is one of the hallmarks of cancer. Ahmadzadeh, M. et al., Blood, 114: 1537-44, disclosed that tumor-specific T lymphocytes are often present in the tumor microenvironment, draining lymph nodes, and peripheral blood, but due to the network of immunosuppressive mechanisms existing in the tumor microenvironment, their The progression of the tumor is often not controlled. CD8 ⁺ tumor-infiltrating T lymphocytes (TILs) typically express activation-induced inhibitory receptors, including CTLA-4 and PD-1, while tumor cells frequently express immunosuppressive ligands, including PD-1 ligand 1 (PD-L1, Also called B7-H1 or CD274), this ligand inhibits T cell activation and effector function. In the inhibitory mechanism, PD-1 and its ligands have become an important way for tumor cells to use it to suppress activated T cells in the tumor microenvironment.

Programmed death receptor 1 (PD-1) plays an important role in immune regulation and peripheral tolerance maintenance. PD-1 is mainly expressed in activated T cells and B cells, and its function is to inhibit the activation of lymphocytes, which is a normal peripheral tissue tolerance mechanism of the immune system to prevent and treat immune overstimulation. However, activated T cells infiltrating in the tumor microenvironment highly express PD-1 molecules, and inflammatory factors secreted by activated leukocytes will induce tumor cells to highly express PD-1 ligands PD-L1 and PD-L2, resulting in high levels of PD-L1 and PD-L2 in the tumor microenvironment. The PD-1 pathway of activated T cells is continuously activated, and the function of T cells is inhibited and cannot kill tumor cells. Therapeutic PD-1 antibodies can block this pathway and partially restore T cell function, allowing activated T cells to continue killing tumor cells.

For nearly a decade, PD-1/PD-L1 pathway blockade has been shown to be an effective way to induce durable antitumor responses in various cancer indications. Monoclonal antibodies (mAbs) that block the PD/PD-L1 pathway can enhance tumor-specific T cell activation and effector function, reduce tumor burden, and improve survival.

Esophageal cancer (EC) is one of the most common malignancies in humans, and its morbidity and mortality have continued to rise over the past few decades, with 400,000 people dying from the disease every year worldwide. Among them, esophageal squamous cell carcinoma (ESCC) is the most common histological subtype of esophageal cancer in developing countries, and it is the predominant histological subtype of esophageal cancer in South American and East Asian populations. Unmet need for treatment. In China, EC is the third most common cancer and the fourth leading cause of cancer-related death. ESCC accounts for more than 90% of all esophageal cancers in China, and is often treated with chemotherapy and radiotherapy. The most commonly used chemotherapy drugs for metastatic ESCC are cisplatin, 5-fluorouracil and taxanes. And the 5-year survival rate of ESCC patients is very low, only 15%-20%. There is currently no standard therapy for patients with chemotherapy-refractory EC. Recently, Clin Cancer Res 2018;24(6):1296-304 disclosed that the anti-PD-1 antibody SHR-1210 has controllable safety and effective antitumor activity in the treatment of chemotherapy-refractory ESCC patients in China, and found that PD -L1-positive tumors had higher objective response rates than PD-L1-negative tumors, and mutational burden (TMB) and potentially the number of mutation-associated neoantigens were found to be associated with better treatment response. In J Clin Oncol 2019; 37, 2019 (suppl 4; abstr 2), it was disclosed that advanced ESCC patients with disease progression after first-line therapy were treated with pembrolizumab in the PD-L1 binding positive score (CPS) ≥ 10 population had a significant overall survival rate compared with chemotherapy. Therefore, although previous studies have demonstrated the efficacy of PD-1-targeted therapy in patients with metastatic ESCC subgroups, predictive biomarkers for the efficacy of anti-PD-1 antibody immunotherapy remain unclear.

SUMMARY OF THE INVENTION

The first aspect of the present invention provides the use of administering an anti-PD-1 antibody or an antigen-binding fragment thereof alone, or in combination with other anticancer agents, in the preparation of a medicament for treating cancer.

In one or more embodiments, other anticancer agents described in the present invention are small molecule targeted anticancer agents. In one embodiment, the other anticancer agents of the present invention are selected from CDK4/6 inhibitors, FGF/FGFR inhibitors.

In one or more embodiments, the medicament comprises an anti-PD-1 antibody or antigen-binding fragment thereof and a CDK4/6 inhibitor. In one or more embodiments, the medicament comprises an anti-PD-1 antibody or antigen-binding fragment thereof and an FGF/FGFR inhibitor.

In one or more embodiments, the present invention provides the use of an anti-PD-1 antibody or antigen-binding fragment thereof for the manufacture of a medicament for the treatment of cancer patients in combination with a CDK4/6 inhibitor, a FGF/FGFR inhibitor.

In one or more embodiments, the present invention provides the use of an anti-PD-1 antibody or antigen-binding fragment thereof in combination with a CDK4/6 inhibitor, a FGF/FGFR inhibitor, for the manufacture of a medicament for the treatment of cancer patients.

In one or more embodiments, the cancer of the present invention is a solid tumor.

In one or more embodiments, the cancer includes, but is not limited to, gastric cancer, esophageal cancer, nasopharyngeal cancer, head and neck squamous cell carcinoma, breast cancer, bladder cancer.

In one or more embodiments, the cancer is preferably esophageal cancer. In one or more preferred embodiments, the cancer is esophageal squamous cell carcinoma. In one or more preferred embodiments, the cancer is advanced ESCC. In one or more preferred embodiments, the cancer is chemotherapy-refractory ESCC. In other embodiments, the esophageal squamous cell carcinoma is advanced, metastatic and/or refractory esophageal squamous cell carcinoma.

In one or more embodiments, the solid tumor does not have gene amplification of the chromosome 11q13 region.

In certain embodiments, the individual has been pretreated. In certain embodiments, the pretreatment includes, but is not limited to, chemotherapy or radiation therapy. In certain embodiments, the individual has undergone systemic therapy. In one or more preferred embodiments, the pre-treatment or systemic treatment is at least 2 times. In certain embodiments, the individual has no history of autoimmune disease or a history of autoimmune disease. In certain embodiments, the individual does not have any concomitant diseases requiring long-term immunosuppressive drug therapy. In certain embodiments, the individual has not been previously treated with any immune checkpoint blockade.

In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is administered alone, or in combination with other anticancer agents, to induce a durable clinical response in the individual.

In one or more embodiments, where the anti-PD-1 antibody or antigen-binding fragment thereof is administered alone, or in combination with other anticancer agents, the therapeutically effective dose of the anti-PD-1 antibody or antigen-binding fragment thereof ranges from About 0.1 to about 10.0 mg/kg body weight by intravenous infusion about every 2 weeks. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is administered at a fixed dose of about 1 mg/kg body weight, 3 mg/kg body weight or 10 mg/kg body weight once every 2 weeks.

A second aspect of the present invention provides a kit for treating an individual with cancer, the kit comprising: (a) a monoclonal antibody or an antigen-binding fragment thereof that specifically binds and inhibits PD-1, and optionally anti-antibody Anti-cancer agents other than PD-1 antibodies or antigen-binding fragments thereof; and (b) administration of anti-PD-1 antibodies or antigen-binding fragments thereof alone, or combined administration of monoclonal antibodies or their antigens that specifically bind and inhibit PD-1 Instructions for combining fragments and other anticancer agents to treat cancer in an individual. The cancer is a solid tumor. As a preferred embodiment, the cancer includes but is not limited to gastric cancer, esophageal cancer, nasopharyngeal cancer, head and neck squamous cell carcinoma, breast cancer, and bladder cancer. As a preferred embodiment, the cancer is preferably esophageal cancer. As a preferred embodiment, the cancer is esophageal squamous cell carcinoma. As a further preferred mode, the cancer is ESCC. As a preferred embodiment, the solid tumor does not have gene amplification in the chromosome 11q13 region.

In one or more embodiments, other anticancer agents described in the present invention are small molecule targeted anticancer agents. In one embodiment, the other anticancer agent of the present invention is selected from one or more of CDK4/6 inhibitors and FGF/FGFR inhibitors.

In one or more embodiments, the kit contains an anti-PD-1 antibody or antigen-binding fragment thereof and a CDK4/6 inhibitor. In one or more embodiments, the kit contains an anti-PD-1 antibody or antigen-binding fragment thereof and an FGF/FGFR inhibitor.

A third aspect of the present invention provides use of a CDK4/6 inhibitor or FGF/FGFR inhibitor in the preparation of a medicament for treating cancer.

A fourth aspect of the present invention provides a method for cancer treatment, comprising the step of sequencing an individual prior to treatment; wherein, an anti-PD-1 antibody or antigen-binding thereof is administered alone to an individual who does not have an amplification of the chromosome 11q13 region Fragments, optionally administered in combination with a CDK4/6 inhibitor, and/or a combination of one or more FGF/FGFR inhibitors; administering a CDK4/6 inhibitor alone, and/or to individuals with amplification of the chromosome 11q13 region Or a combination of one or more FGF/FGFR inhibitors, optionally an anti-PD-1 antibody or an antigen-binding fragment thereof is administered in combination.

In the uses, methods, medicaments and kits of the present invention, the individual is a human.

In one or more preferred embodiments, the subject of the present invention is a human and his cancer is a solid tumor. In one or more preferred embodiments, the subject of the present invention is a human and his cancer is selected from the group consisting of, but not limited to, gastric cancer, esophageal cancer, nasopharyngeal cancer, head and neck squamous cell carcinoma, breast cancer, bladder cancer cancer. In a preferred embodiment, the subject of the present invention is a human and the cancer is esophageal cancer. In a preferred embodiment, the subject of the present invention is a human and his cancer is ESCC. In one or more embodiments, the individual described herein has esophageal cancer and has not received prior immunotherapy.

A fourth aspect of the present invention provides a method for predicting the effect of anti-PD-1 antibody therapy in a tumor patient, comprising detecting whether the patient has gene amplification in the chromosome 11q13 region, wherein the absence of the gene amplification in the chromosome 11q13 region indicates that the The tumor patients described above are suitable for treatment with anti-PD-1 antibodies.

In the uses, methods, medicines and kits of the present invention, the anti-PD-1 antibody is a monoclonal antibody or an antigen-binding fragment thereof. In certain embodiments, the anti-PD-1 antibody specifically binds PD-1 and blocks the binding of PD-L1 or PD-L2 to PD-1. In certain embodiments, the anti-PD-1 antibody specifically binds PD-L1 or/and PD-L2, blocking the binding of PD-L1 and/or PD-L2 to PD-1.

In one or more embodiments, the anti-PD-1 antibody is an antibody comprising a complementarity determining region (CDR) with a light chain complementarity determining region (LCDR) as set forth in SEQ ID NOs: 1, 2 and 3, a heavy chain complementary The determining regions (HCDRs) are shown in SEQ ID NOs: 4, 5 and 6.

In one or more embodiments, the anti-PD-1 antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein VL is set forth in SEQ ID NO: 7 and VH is set forth in SEQ ID NO : shown in 8.

In one or more embodiments, the anti-PD-1 antibody is an anti-PD-1 antibody comprising a light chain and a heavy chain, and the light chain comprises the amino acid sequence set forth in SEQ ID NO:9 and the heavy chain comprises SEQ ID NO: The amino acid sequence shown in 10.

Description of drawings

Figure 1: (A) Maximum change from baseline in tumor size as assessed by investigators according to RECIST v1.1 (n=46, subjects had baseline and at least one post-treatment imaging assessment). The length of the bar graph represents the maximal reduction or minimal increase in the target lesion. The colors of the bar graphs reflect the results of previous systemic treatments. Blue: ≥3L; Orange: 2L; Green: 1L. Five patients (marked with #) had the best change from baseline in target lesions, a >30% decrease, but responses were not confirmed or new lesions developed. Five patients (marked as +) had the best change from baseline in target lesions with a less than 20% increase, but were characterized by progressive disease (PD) due to the occurrence of new lesions or progression of non-target lesions.

(B) Investigators assessed individual tumor burden over time per RECIST v1.1 (n=46, subject baseline and at least 1 post-treatment radiograph). *Change percentage is truncated to 100%.

(C) Progression-free survival for all patients in the study.

(D) Overall survival for all patients in the study. The percentage of surviving patients at the indicated time points is shown. The patients under review are marked with "┃" in the figure. The number of patients at risk at a given time point is shown below the x-axis.

Figure 2: Clinical response associated with tumor PD-L1 expression or tumor mutational burden (TMB).

(A) PD-L1 positive status was defined as the presence of ≥1% intensity of membrane staining of tumor cells or immune cells stained by SP142 containing IHC.

(B) TMB status was determined by whole-exome sequencing of tumor biopsies and paired PBMCs.

(C) PFS comparison of PD-L1-positive versus PD-L1-negative patients.

(D) Comparison of OS in PD-L1-positive versus PD-L1-negative patients.

(E) Comparison of PFS in patients with high and low TMB.

(F) Comparison of OS in patients with high TMB (≥12) and low TMB (<12). The percentage of surviving patients is shown at the indicated time points. The patients under review are marked with "┃" in the picture. The number of patients at risk at the indicated time point is shown below the x-axis.

Figure 3: Mutation profile of patients with advanced EC.

Genomic profiling of WES revealed that missense mutations or truncations in TP53 (p53) (76%), RYR2 (22%), NOTCH1 (20%), LRP1B (17%), and TRIO (17%) were the most common tumor biopsies in ESCC 5 common mutated genes.

Figure 4: 11q13 locus amplification and RNA expression analysis of selected ESCC patients.

(A) Genome profiling by next-generation sequencing of FFPE tumor and paired peripheral blood samples from 51 available patients. 11q13 was amplified in 24 cases. At the top is a map of the chromosome 11q13 region and coding genes. Three algorithms are used to determine amplification events. Each individual has an amplified gene. Mutations or deletions of amplified genes are also flagged.

(B) Messenger RNA expression analysis of amplified 11q13. mRNA sequencing and expression profiling were performed on existing patients. mRNA expression levels correlated with gene amplification in each individual.

Figure 5: Clinical response correlates with gene amplification status in chromosome 11q13 region.

(A) Tumor 11q13 status was determined by whole-exome sequencing of tumor biopsies and paired PBMCs.

(B) Progression-free survival of 11q13 wild-type and 11q13-amplified patients.

(C) Overall survival of 11q13 wild-type versus 11q13-amplified patients. The percentage of surviving patients is shown at the indicated time points. The patients under review are marked with "┃" in the picture. The number of patients at risk at the indicated time point is shown below the x-axis.

Figure 6: Clinical trial progress of the NCT02915432 study to evaluate the effect of toripalimab in patients with advanced GC, ESCC, NPC and HNSCC.

Detailed ways

The present invention relates to tumor treatment methods. The methods of the invention comprise administering to a patient in need thereof an anti-PD-1 antibody or antigen-binding fragment thereof alone; or comprising administering to a patient in need thereof an anti-PD-1 antibody or antigen-binding fragment thereof in combination with other anticancer agents. The present invention also relates to a method for predicting the efficacy of anti-PD-1 antibodies in the treatment of cancer, especially in patients with esophageal cancer, using biomarkers.

the term

For easier understanding of the present invention, certain scientific and technical terms are specifically defined below. Unless clearly defined otherwise herein, technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

"Administering," "administering," and "treating" refer to introducing a composition comprising a therapeutic agent into a subject using any of a variety of methods or delivery systems known to those of skill in the art. Routes of administration of anti-PD-1 antibodies include intravenous, intramuscular, subcutaneous, peritoneal, spinal or other parenteral routes of administration, such as injection or infusion. "Parenteral administration" refers to modes of administration other than enteral or topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular , intra-frame, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, intradural, and intrasternal injection and infusion and in vivo electroporation.

An "adverse effect" (AE) as described herein is any unfavorable and often unintended or undesired sign, symptom or disease associated with the use of a medical treatment. For example, adverse effects may be related to activation of the immune system or expansion of immune system cells in response to treatment. A medical treatment may have one or more associated AEs, and each AE may be of the same or a different level of severity.

The term "subject" includes any organism, preferably an animal, more preferably a mammal (eg, rat, mouse, dog, cat, rabbit, etc.), and most preferably a human. The terms "subject", "patient" and "individual" are used interchangeably herein.

"Antibody" as used herein refers to any form of antibody capable of achieving the desired biological or binding activity. Thus, it is used in the broadest sense, but is not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies, humanized full-length human antibodies, chimeric antibodies, and camel-derived single domain antibodies. It specifically binds antigen and comprises at least two heavy (H) and two light (L) chains interconnected by disulfide bonds, or antigen-binding fragments thereof. Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region comprising three constant domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (VL) and a light chain constant region, the light chain constant region comprising a constant domain CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). In general, both the light and heavy chain variable domains comprise FRl, CDRl, FR2, CDR2, FR3, CDR3 and FR4 from the N-terminus to the C-terminus. Amino acids are generally assigned to each domain according to the following definitions: Sequences of Proteins of Immunological Interest, Kabat et al; National Institutes of Health, Bethesda, Md.; 5th Edition; NIH Publication No. 91-3242 (1991): Kabat (1978) Adv. Prot. Chem. 32:1-75; Kabat et al, (1977) J. Biol. Chem. 252:6609-6616; Chothia et al, (1987) J Mol. Biol. 196:901 -917 or Chothia et al. (1989) Nature 341:878-883.

The carboxy-terminal portion of the heavy chain may define the constant region primarily responsible for effector function. Generally, human light chains are classified into κ chains and λ chains. Human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG subclasses are well known to those of skill in the art and include, but are not limited to, IgGl, IgG2, IgG, and IgG4.

The term "antibody" includes: naturally occurring and non-naturally occurring Abs; monoclonal and polyclonal Abs; chimeric and humanized Abs; human or non-human Abs; fully synthetic Abs; and single chain Abs. Non-human Abs can be humanized by recombinant methods to reduce their immunogenicity in humans.

Unless expressly stated otherwise, an "antibody fragment" or "antigen-binding fragment" as used herein refers to an antigen-binding fragment of an antibody, ie, an antibody fragment that retains the ability of a full-length antibody to specifically bind to an antigen, such as retaining one or more Fragments of multiple CDR regions. Examples of antibody-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; nanobodies, and multispecific antibodies formed from antibody fragments.

"Chimeric antibody" refers to antibodies and fragments thereof in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in an antibody derived from a particular species (eg, human) or belonging to a particular antibody class or subclass , while the remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species (eg, mouse) or belonging to another antibody class or subclass, so long as it exhibits the desired biological activity.

"Human antibody" refers to an antibody comprising only human immunoglobulin sequences. If the human antibody is produced in a mouse, a mouse cell, or a mouse cell-derived hybridoma, it may contain a murine carbohydrate chain. Similarly, "mouse antibody" or "rat antibody" refers to an antibody comprising only mouse or rat immunoglobulin sequences, respectively.

"Humanized antibody" refers to a form of antibody that contains sequences from non-human (eg, murine) antibodies as well as from human antibodies. Such antibodies contain minimal sequence derived from the unilateral side of the non-human immunoglobulin. Typically, a humanized antibody will comprise substantially all of at least one and usually both variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of the non-human immunoglobulin, and all or substantially all The FR regions are those of human immunoglobulins. The humanized antibody optionally also includes at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin constant region.

As used herein, the term "cancer" refers to a wide variety of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division, growth division and growth leads to the formation of malignant tumors that invade adjacent tissues and can also metastasize to distant parts of the body through the lymphatic system or bloodstream. Examples of cancer include, but are not limited to, carcinoma, lymphoma, leukemia, blastoma, and sarcoma. More specific examples of cancer include squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer, glioma, Hodgkin lymphoma, non-Hodgkin lymphoma, acute myeloid leukemia, multiple myeloma , gastrointestinal (tract) cancer, kidney cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, Neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, brain cancer, stomach cancer, bladder cancer, hepatocellular tumor, breast cancer, colon cancer, and head and neck cancer. Another embodiment of cancer includes esophageal cancer. Another specific example of cancer includes ESCC. Another specific example of cancer includes chemotherapy-refractory ESCC. Another specific example of cancer includes advanced ESCC. In certain embodiments, cancers described herein include those cancers characterized by a sequenced individual having gene amplification in the 11q13 region of chromosome. Cancers of the present invention include those characterized by the absence of gene amplification in the 11q13 region of chromosomes in the sequenced individual.

The term "esophageal cancer" or "EC" is one of the most common malignant tumors in humans, which is a common gastrointestinal tumor and can be divided into squamous cell carcinoma, adenocarcinoma and small cell carcinoma according to histological and pathological types three. Among them, "esophageal squamous cell carcinoma" or "ESCC" is the most common type of EC in developing countries, and the prognosis of this cancer is poor, with a 5-year overall survival rate of only 20-30%.

The term "immunotherapy" refers to the treatment of a subject suffering from a disease or at risk of infection or recurrence of the disease by methods that include inducing, enhancing, inhibiting or otherwise modifying an immune response. "Treatment" or "therapy" of a subject refers to any type of intervention or procedure performed on a subject, or administration of an active agent to the subject, for the purpose of reversing, alleviating, ameliorating, alleviating or preventing symptoms, complications or the onset, progression, severity or recurrence of a condition, or a biochemical marker associated with the disease.

"Programmed death receptor-1 (PD-1)" refers to an immunosuppressive receptor belonging to the CD28 family. PD-1 is mainly expressed on previously activated T cells in vivo and binds two ligands, PD-L1 and PD-L2. The term "PD-1" as used herein includes human PD-1 (hPD-1), variants, isoforms and species homologues of hPD-1, and analogs that share at least one epitope with hPD-1 .

A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is any amount of a drug that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of disease or promotes regression of disease, the Disease regression is evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of the disease, or prevention of injury or disability caused by the suffering of the disease. The ability of a therapeutic agent to promote disease regression can be assessed using a variety of methods known to those of skill in the art, such as in human subjects during clinical trials, in animal model systems that predict efficacy in humans, or by in vitro assays The activity of the agent was determined.

A therapeutically effective amount includes a "prophylactically effective amount," that is, any amount that inhibits the development or recurrence of cancer when administered alone or in combination with an anti-tumor agent to a subject at risk of developing cancer or to a subject suffering from cancer recurrence medicine.

"Biotherapeutics" refers to biomolecules, such as antibodies or fusion proteins, that block ligand/receptor signaling in any biological pathway that supports tumor maintenance and/or growth or inhibits an anti-tumor immune response.

Unless expressly stated otherwise, as used herein, "CDRs" refer to immunoglobulin variable regions that are complementarity determining regions as defined using the Kabat numbering system.

"Anticancer agent" refers to any therapeutic agent that can be used to treat cancer. Classes of anticancer agents include, but are not limited to: alkylating agents, antimetabolites, kinase inhibitors, spindle poison phytoalkaloids, cytotoxic/antineoplastic antibiotics, photosensitizers, antiestrogens, and selective estrogen receptor modulators Anti-progesterone agents, aromatase inhibitors, CDK4/6 inhibitors, FGF/FGFR inhibitors, etc., and antisense oligonucleotides that inhibit the expression of genes involved in abnormal cell proliferation or tumor growth.

The term "about" refers to a value or composition within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which depends in part on how the value or composition is measured or determined, ie, the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation according to practice in the art. Alternatively, "about" may refer to a range of up to 10% or 20% (ie, ±10% or ±20%). For example, about 3 mg/kg can include any number between 2.7 mg/kg and 3.3 mg/kg (relative to 10%), and between 2.4 mg/kg and 3.6 mg/kg (relative to 20%). Herein, when a particular value or composition is provided, unless expressly stated otherwise, the meaning of "about" shall be assumed to be within an acceptable error range for that particular value or composition.

"Therapeutic anti-PD-1 monoclonal antibody" refers to an antibody that specifically binds to a specific mature form of PD-1 expressed on the surface of certain mammalian cells. Mature PD-1 has no prosecretory leader sequence, or leader peptide. The terms "PD-1" and "mature PD-1" are used interchangeably herein and are to be understood to mean the same molecule unless expressly defined otherwise, or clear from the context.

As used herein, a therapeutic anti-human PD-1 antibody or anti-hPD-1 antibody refers to a monoclonal antibody that specifically binds to mature human PD-1.

"Framework regions" or "FRs" as used herein refer to immunoglobulin variable regions that do not include CDR regions.

"Isolated antibody or antigen-binding fragment thereof" refers to a purified state and in which case the designated molecule is substantially free of other biological molecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials such as cellular debris or growth culture medium).

"Patient," "patient," or "subject" refers to any single subject, including humans and mammals, such as horses, cattle, and dogs, in need of medical procedures or participation in clinical trials, epidemiological studies, or use as controls or cat.

In the following paragraphs, various aspects of the invention are described in further detail.

anti-PD-1 antibody

"PD-1 antibody" refers to binding to the PD-1 receptor, blocking the binding of PD-L1 expressed on cancer cells to PD-1 expressed on immune cells (T, B, NK cells) and preferably also blocking Any chemical compound or biomolecule that blocks the binding of PD-L2 expressed on cancer cells to PD-1 expressed on immune cells. Alternative nouns or synonyms for PD-1 and its ligands include: PDCD1, PD1, CD279 and SLEB2 for PD-1; PDCD1L1, PDL1, B7-H1, B7H1, B7-4, CD274 and B7-H; and for PD-L2 PDCD1L2, PDL2, B7-DC and CD273. In any of the therapeutic methods, medicaments and uses of the invention for treating a human subject, the PD-1 antibody blocks the binding of human PD-L1 to human PD-1, and preferably blocks both human PD-L1 and PD-L2 from binding to human PD1 binding. The human PD-1 amino acid sequence can be found at NCBI locus number: NP_005009. Human PD-L1 and PD-L2 amino acid sequences can be found at NCBI locus numbers: NP_054862 and NP_079515, respectively.

Anti-PD-1 antibodies useful in any of the uses, therapies, medicaments and kits described herein include monoclonal antibodies (mAbs) or antigen-binding fragments thereof that specifically bind to PD-1, and preferably specifically bind to Human PD-1. mAbs can be human, humanized, or chimeric antibodies, and can include human constant regions. In some embodiments, the constant region is selected from the group consisting of human IgGl, IgG2, IgG3, and IgG4 constant regions, and in preferred embodiments, the constant region is a human IgG4 constant region.

In any one of the embodiments of the uses, therapies, medicaments and kits of the present invention, the PD-1 antibody is a monoclonal antibody or an antigen-binding fragment thereof, comprising: (a) the light chain CDR is SEQ ID NO: 1 The amino acids shown in , 2 and 3, and the heavy chain CDRs are the amino acids shown in SEQ ID NOs: 4, 5 and 6.

In any one of the embodiments of the uses, therapies, medicaments and kits described herein, the PD-1 antibody specifically binds to human PD-1 and comprises: (a) a light chain comprising SEQ ID NO:7 can variable region, and (b) a monoclonal antibody comprising the heavy chain variable region of SEQ ID NO:8.

In any one of the embodiments of the uses, therapies, medicaments and kits described herein, the PD-1 antibody specifically binds to human PD-1 and comprises: (a) a light chain comprising SEQ ID NO:9, and (b) a monoclonal antibody comprising the heavy chain of SEQ ID NO:10.

In any one of the embodiments of the uses, therapies, medicaments and kits of the present invention, the PD-1 antibody is a monoclonal antibody or an antigen-binding fragment thereof, and Table A below provides the uses, therapies, and treatments of the present invention. List of amino acid sequences of exemplary anti-PD-1 antibody mAbs in , drugs and kits:

Table A: Light and heavy chain CDRs of exemplary anti-human PD-1 antibodies

CDRL1 SEQ ID NO: 1 CDRL2 SEQ ID NO: 2 CDRL3 SEQ ID NO: 3 CDRH1 SEQ ID NO: 4 CDRH2 SEQ ID NO: 5 CDRH3 SEQ ID NO: 6

Examples of anti-PD-1 antibodies that bind to human PD-1 and can be used in the uses, therapies, medicaments and kits described herein are described in WO2014206107. Human PD-1 mAbs that can be used as anti-PD-1 antibodies in the uses, therapies, medicines and kits of the present invention include any one of the anti-PD-1 antibodies described in WO2014206107, including: toripalimab (Toripalimab) (a compound having the structure described in WHO Drug Information (Vol. 32, Issue 2, pp. 372-373 (2018)) and comprising the sequences shown in SEQ ID NOs: 9 and 10 humanized IgG4 mAbs of light and heavy chain amino acid sequences). In certain embodiments, anti-PD-1 antibodies useful in the uses, therapies, medicaments, and kits described herein also include FDA-approved nivolumab and pembrolizumab, and NMPA-approved letters Dilimumab, as well as SHR-1210 and Tislelizumab in clinical stages, and in preclinical and clinical stages can block PD-L1 expressed on cancer cells and immune cells (T, B, NK cells) Any chemical compound or biomolecule that binds to PD-1 on ) and preferably also blocks the binding of PD-L2 expressed on cancer cells to PD-1 expressed on immune cells.

In certain embodiments, anti-PD-1 antibodies useful in the uses, therapies, medicaments and kits described herein also include anti-PD antibodies that specifically bind to PD-L1 to block the binding of PD-L1 to PD-1 -L1 monoclonal antibody, such as atezolizumab, avelumab, durvalumab, or any chemical compound or biomolecule that can specifically bind to PD-L1 to block the binding of PD-L1 to PD-1.

"PD-L1" expression or "PD-L2" expression as used herein refers to any detectable level of expression of a specific PD-L protein on a cell surface or a specific PD-L mRNA within a cell or tissue. PD-L protein expression can be detected using diagnostic PD-L antibodies in IHC analysis of tumor tissue sections or by flow cytometry. Alternatively, PD-L protein expression by tumor cells can be detected by PET imaging using a binding agent that specifically binds to a desired PD-L target, such as PD-L1 or PD-L2.

Methods for quantifying PD-L1 protein expression in IHC analysis of tumor tissue sections can be found in, but not limited to, Thompson, R.H. et al., PNAS 101(49):17174-17179 (2004); Taube, J.M. et al., SciTransl Med 4, 127ra37 (2012); and Toplian, S.L. et al., New Eng. J. Med. 366(26):2443-2454 (2012) et al.

One approach employed a simple binary endpoint of positive or negative PD-L1 expression, where a positive result for PD-L1 expression was defined by the percentage of tumor cells showing histological evidence of cell surface membrane staining. Tumor tissue sections counted as at least 1% of total tumor cells were defined as positive for PD-L1 expression.

In another method, PD-L1 expression in tumor tissue sections is quantified in tumor cells as well as in infiltrating immune cells. The percentages of tumor cells and infiltrating immune cells exhibiting membrane staining were individually quantified as <1%, 1% to 50%, and then 50% up to 100%. For tumor cells, PD-L1 expression was counted as negative if the score was <1%, and positive if the score was ≥1%.

In some embodiments of the invention, the PD-L1 expression in the subject's tumor tissue section is ≥ 1%, compared to the PD-L1 expression in the subject's tumor tissue section < 1%, no comparison is obtained good benefit. In some preferred embodiments, the cancer is a solid tumor. In some specific embodiments, the cancer is esophageal cancer. As a preferred embodiment, the cancer is ESCC.

"RECIST 1.1 Efficacy Criteria" as described herein refers to the definitions described by Eisenhauver et al., E.A. et al., Eur. J Cancer 45:228-247 (2009) for target injury or non-target injury based on the context of the measured response.

The term "ECOG" scoring scale is a measure of a patient's general health and tolerance to treatment based on their physical strength. ECOG physical condition scoring standard scoring: 0 points, 1 point, 2 points, 3 points, 4 points and 5 points. A score of 0 is defined as completely normal activity without any difference from pre-onset activity. A score of 1 was defined as being able to walk freely and engage in light physical activity, including general housework or office work, but not in heavier physical activity.

A "sustained response" refers to a sustained therapeutic effect after discontinuation of the therapeutic agent or combination therapy described herein. In some embodiments, the sustained response has a duration at least the same as or at least 1.5, 2.0, 2.5 or 3 times the duration of treatment.

"Tissue section" refers to a single portion or slice of a tissue sample, such as a tissue slice cut from a sample of normal tissue or tumor.

"Treatment" of cancer as described herein refers to the use of a treatment regimen described herein (eg, administration of an anti-PD-1 antibody, or administration of an anti-PD-1 antibody in combination with CDK4/6 inhibition in a subject having or diagnosed with cancer) as described herein agent or combination therapy with an FGF/FGFR inhibitor) to achieve at least one positive therapeutic effect (e.g., reduction in cancer cell number, reduction in tumor volume, reduction in the rate of cancer cell infiltration into surrounding organs, or reduction in the rate of tumor metastasis or tumor growth) ). Positive therapeutic effects in cancer can be measured in a variety of ways (see W.A. Weber, J. Nucl. Med., 50: 1S-10S (2009)). For example, regarding tumor growth inhibition, according to NCI criteria, T/C≦42% is the minimum level of antitumor activity. Consider T/C (%) = median treated tumor volume/median control tumor volume x 100. In some embodiments, the therapeutic effect achieved by the combination of the present invention is any of PR, CR, OR, PFS, DFS and OS. PFS (also called "time to tumor progression") refers to the length of time during and after treatment that the cancer does not grow, and includes the amount of time a patient experiences CR or PR as well as the amount of time a patient experiences SD. DFS refers to the length of time a patient remains disease free during and after treatment. OS refers to an increase in life expectancy compared to an initial or untreated individual or patient. In some embodiments, the response to the combination of the invention is any of PR, CR, PFS, DFS, OR, or OS, as assessed using the RECIST 1.1 efficacy criteria. Treatment regimens for the combinations of the invention that are effective in treating cancer patients can vary depending on factors such as the patient's disease state, age, weight, and the ability of the therapy to elicit an anticancer response in the subject. Although embodiments of the present invention may not achieve an effective positive therapeutic effect in every subject, they should be effective and achieve a positive therapeutic effect in a statistically significant number of subjects.

The terms "administration mode" and "dosing regimen" are used interchangeably and refer to the dosage and timing of use of each therapeutic agent in the combination of the present invention.

"Tumor" as applied to a subject diagnosed with or suspected of having cancer refers to a malignant or potentially malignant neoplasm or mass of tissue of any size, and includes both primary tumors and secondary neoplasms. Solid tumors usually do not contain cysts or abnormal growths or masses of tissue in areas of fluid. Different types of solid tumors are named for the type of cells that form them. Examples of solid tumors are sarcomas, carcinomas and lymphomas. Blood cancers do not usually form solid tumors.

"Tumor burden" refers to the total amount of tumor material distributed throughout the body. Tumor burden refers to the total number of cancer cells or the total size of a tumor throughout the body. Tumor burden can be determined by a variety of methods known in the art, such as the use of calipers after the tumor is removed from the subject, or imaging techniques (eg, ultrasound, bone scan, computed tomography) when in vivo (CT) or Magnetic Resonance Imaging (MRI) scans) to measure its size.

The term "tumor size" refers to the total size of the tumor, which can be measured as the length and width of the tumor. Tumor size can be determined by a variety of methods known in the art, such as using calipers after the tumor is removed from the subject, or using imaging techniques such as bone scans, ultrasound, CT or MRI scans when in vivo. size.

The term "tumor mutational burden (TMB)" refers to the total number of somatic gene coding errors, base substitutions, gene insertions or deletions errors detected per megabase. In some embodiments of the invention, tumor mutational burden (TMB) is estimated by analyzing somatic mutations including coding base substitutions and megabase insertions of the panel sequences studied. In some embodiments of the present invention, the subject's tumor mutational burden (TMB) is greater than or equal to 12 mutations/Mb, which is not better than the subject's tumor mutational burden TMB<12 mutations/Mb benefit. In some preferred embodiments, the cancer is a solid tumor. In some specific embodiments, the cancer is esophageal cancer. As a preferred embodiment, the cancer is ESCC.

The term "gene amplification" refers to the process by which the copy number of a gene encoding a specific protein is selectively increased while other genes are not proportionally increased. Under natural conditions, gene amplification is achieved by excision of repeats of the gene from chromosomes and extrachromosomal replication in plasmids or by transcribing the entire repeats of ribosomal RNA into RNA transcripts and then transcribing additional copies of the original DNA molecule of. In the present invention, gene sequencing analysis is disclosed in some embodiments. In some embodiments of the invention, the subject of the invention has certain unique gene amplifications. In some preferred embodiments, the subject has a gene amplification of the 11q13 region of chromosome. In still further preferred embodiments, the subject has CDK4/6 gene amplification; in still further preferred embodiments, the subject has FGF3/4/19 gene amplification. As a preferred embodiment, the esophageal cancer subject has CDK4/6 gene amplification. As a preferred embodiment, the esophageal cancer subject has FGF3/4/19 gene amplification. In some embodiments, the tumor patient has gene amplification in the 11q13 region of chromosome, which is predictive of the availability of (a) CDK4/6 inhibitor and/or FGF/FGFR inhibitor administered alone, or (b) in combination with The therapeutic effect of PD-1 antibody or its antigen-binding fragment in combination with one or more of CDK4/6 inhibitor and/or FGF/FGFR inhibitor is better. In some embodiments, the tumor patient does not have gene amplification in the 11q13 region of chromosome, indicating that he can be administered (a) an anti-PD-1 antibody or antigen-binding fragment thereof alone, or (b) a combination of anti-PD-1 1. The therapeutic effect of the combination of the antibody or its antigen-binding fragment and one or more of the CDK4/6 inhibitor and/or the FGF/FGFR inhibitor is better.

The term "CDK" stands for cyclin-dependent kinases, which is a group of serine/threonine protein kinases. CDKs drive the cell cycle through phosphorylation of serine/threonine proteins in synergy with cyclin. It is an important factor in cell cycle regulation. There are 8 types of CDK family including CDK 1-8, each CDK binds to different types of cyclins to form complexes, and regulates the process of cells transitioning from G1 phase to S phase or G2 phase to M phase and exiting M phase. At present, the CDK4/6 inhibitors that have been approved by the FDA mainly include ribociclib and palbociclib, and there are dozens of CDK4/6 inhibitors in the clinical research stage.

The term "FGF" refers to the fibroblast growth factor (Fibroblast growth factor) protein family, which has 23 members in total, FGF1-23. According to their different mechanisms of action, FGFs can be divided into three categories: endocrine (FGF15/19/21/23), paracrine (FGF1-10, FGF16-18, FGF20, FGF22) and secretory FGF11/12/13/14 . The process of paracrine FGFs regulating biological activity is to use HS as a cofactor to specifically bind to FGF receptors (Fibroblast Growth Factor Receptor, FGFRs) on the cell surface. The FGFRs mainly include four kinds: FGFR1, FGFR2, FGFR3, FGFR4. There is no FGF/FGFR inhibitor on the market yet. Boehringer Ingelham's VEGFR/PDGFR/FGFR inhibitor nintedanib for the treatment of liver cancer, non-small cell lung cancer, and idiopathic fibrosis was granted breakthrough drug qualification by the FDA in 2014. In China, dalitinib is currently in the clinical stage.

The term "Immunohistochemistry (IHC)" refers to the use of the principle of specific binding of antigens and antibodies to determine the intracellular antigens in tissue cells by chemical reaction to develop color reagents (fluorescein, enzymes, metal ions, isotopes) labeled with antibodies. (polypeptides and proteins), and methods for localization, qualitative and relative quantitative research. In some embodiments of the invention, prior to treatment with an anti-PD-1 antibody, the subject's tumor tissue sample is subjected to PD-L1 expression detection using Roche's anti-human PD-L1 antibody SP142 (Cat No: M4422) for staining experiments. In some embodiments, tumor cells are defined as PD-L1 positivity by membrane staining intensity > 1%.

Pharmaceutical composition and dosage

The therapeutic agent of the present invention can constitute a pharmaceutical composition, such as a pharmaceutical composition containing the anti-PD-1 antibody described herein or/and other anti-cancer agents other than the anti-PD-1 antibody and other pharmaceutically acceptable carriers . As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, carriers suitable for use in compositions containing anti-PD-1 antibodies are suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration, such as by injection or infusion, for use in compositions containing other anticancer agents The carrier of the composition is suitable for parenteral administration, such as oral administration. The pharmaceutical compositions of the present invention may contain one or more pharmaceutically acceptable salts, antioxidants, water, non-aqueous carriers, and/or adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents.

Dosage regimens are adjusted to provide the best desired response, such as maximal therapeutic response and/or minimal adverse effects. For anti-PD-1 antibodies, including administration in combination with another anticancer agent, the dose range may be from about 0.01 to about 20 mg/kg, from about 0.1 to about 10 mg/kg of individual body weight, or a fixed 120 mg, 240 mg, 360 mg, 480 mg dose. For example, the dose can be about 0.1, about 0.3, about 1, about 2, about 3, about 5, or about 10 mg/kg of the subject's body weight. Dosage regimens are typically designed to achieve exposures that result in sustained receptor occupancy (RO) based on the Ab's typical pharmacokinetic properties. A representative dosing regimen may be about once a week, about once every two weeks, about once every three weeks, about once every four weeks, about once a month, or more. In some embodiments, the anti-PD-1 antibody is administered to the individual about once every two weeks.

Dosing schedules for other anticancer agents vary from drug to drug.

In some embodiments of the invention, the dosing schedule of the CDK4/6 inhibitor or FGF/FGFR inhibitor varies for different subtypes. For combination therapy with an anti-PD-1 antibody and a CDK4/6 inhibitor or FGF/FGFR inhibitor, in some embodiments, the CDK4/6 inhibitor or FGF/FGFR inhibitor is administered at its approved or recommended dose, continuing Treat until clinical effect is observed or until unacceptable toxicity or disease progression occurs.

Method of the present invention

The present invention relates to a method of treating a cancer patient comprising administering to the cancer patient (a) a therapeutically effective amount of an anti-PD-1 antibody or antigen-binding fragment thereof alone, or (b) in combination with a therapeutically effective amount of an anti-PD-1 antibody or its A combination of one or more of the antigen-binding fragments and optionally other anti-cancer agents other than anti-PD-1 antibodies and antigen-binding fragments thereof.

In certain embodiments, as described herein, (a) a therapeutically effective amount of an anti-PD-1 antibody or antigen-binding fragment thereof is administered alone, or (b) a therapeutically effective amount of an anti-PD-1 antibody is administered in combination, or Cancer patients treated with the combination of one or more of its antigen-binding fragments and optionally other anti-cancer agents other than anti-PD-1 antibodies and its antigen-binding fragments are preferably cancers with no detectable gene amplification in the chromosome 11q13 region patient. Accordingly, in some embodiments, the cancer treatment method further comprises the step of sequencing the cancer patient.

In certain embodiments, the present invention relates to a method of treating a cancer patient comprising administering to the cancer patient (a) a therapeutically effective amount of another anticancer agent alone, or (b) in combination with a therapeutically effective amount of an anti-PD-1 antibody or A combination of one or more of its antigen-binding fragments and optionally other anti-cancer agents other than anti-PD-1 antibodies.

In certain embodiments, the invention is suitable for (a) single administration of a therapeutically effective amount of other anticancer agents, or (b) combined administration of a therapeutically effective amount of an anti-PD-1 antibody or antigen-binding fragment thereof and any Selected cancer patients treated with one or more combinations of other anti-cancer agents other than anti-PD-1 antibodies preferably have gene amplification in the chromosome 11q13 region, especially CDK4/6 gene amplification or FGF3/ 4/19 cancer patients with gene amplification. Accordingly, in some embodiments, the cancer treatment method further comprises the step of sequencing the cancer patient.

In certain embodiments, the other anticancer agent is a therapeutically effective amount of a CDK4/6 inhibitor and/or a FGF/FGFR inhibitor.

In certain embodiments, the present invention provides methods of treating individuals/patients with esophageal cancer. In some embodiments, the method comprises administering to the individual a therapeutically effective dose of a combination of: (a) an Ab or antigen-binding fragment thereof that specifically binds to the PD-1 receptor and inhibits PD-1 activity; and (b) Another anticancer therapy. In one embodiment, the method comprises administering to the individual an effective amount of a combination of (i) a standard therapy for the treatment of esophageal cancer as disclosed elsewhere herein, or (ii) other anticancer agents. In some embodiments, the other anticancer agent is selected from CDK4/6 inhibitors and/or FGF/FGFR inhibitors. Since the most common histological subtype of esophageal cancer in patients with esophageal cancer in developing countries is esophageal squamous cell carcinoma (ESCC), in some embodiments, the esophageal cancer is esophageal squamous cell carcinoma (ESCC). . In certain embodiments, the method further comprises, prior to administration of the therapeutic drug, the step of sequencing the patient; according to the sequencing results, administering the anti-PD-1 antibody alone or to the individual who does not have the amplification of the chromosome 11q13 region Antigen-binding fragments thereof, optionally combined with one or more combinations of CDK4/6 inhibitors and/or FGF/FGFR inhibitors; Individuals with increased or FGF3/4/19 gene amplification are administered CDK4/6 inhibitors alone, and/or one or more combinations of FGF/FGFR inhibitors, optionally in combination with anti-PD-1 antibodies or its antigen-binding fragment.

Anti-PD-1 antibodies suitable for use in the methods of the invention

The antibody PD-1 antibody suitable for the method of the present invention binds PD-1 with high specificity and affinity, blocks the binding of PD-L1/2 to PD-1, and inhibits PD-1 signal transduction. Inhibitory effect. In any of the methods of treatment disclosed herein, an anti-PD-1 antibody includes an antigen-binding portion or fragment that binds to the PD-1 receptor and exhibits functional properties similar to that of an intact Ab in terms of inhibiting ligand binding and upregulating the immune system. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is an anti-PD-1 antibody or antigen-binding fragment thereof that cross-competes with toripalimab for binding to human PD-1. In other embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is a chimeric, humanized or human Ab or antigen-binding fragment thereof. In certain embodiments for the treatment of human subjects, the Ab is a humanized Ab.

In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain constant region of human IgG1 or IgG4 isotype. In some embodiments, the sequence of the IgG4 heavy chain constant region of the anti-PD-1 antibody or antigen-binding fragment thereof comprises the S228P mutation that replaces the hinge region with a proline residue normally present at the corresponding position in an antibody of the IgG1 isotype serine residues in . In certain embodiments of any of the methods of treatment described herein comprising administering an anti-PD-1 antibody, the anti-PD-1 antibody is toripalimab. In some embodiments, the anti-PD-1 antibody is selected from the humanized antibodies 38, 39, 41 and 48 described in WO2014206107. In some embodiments, the anti-PD-1 antibody is as described above in the "Anti-PD-1 Antibody" section.

CDK4/6 inhibitors suitable for use in the methods of the invention

In some embodiments of the present invention for the treatment of tumors, the other anti-cancer agent, ie, the anti-cancer agent used in combination with the anti-PD-1 antibody in addition to the anti-PD-1 antibody, or the anti-cancer agent used alone is a CDK4/6 inhibitor. Currently FDA-approved CDK4/6 inhibitors are mainly Eli Lilly's Verzenio (abemaciclib), which is mainly used to treat hormone receptor (HR) positive and human epidermal growth factor receptor 2 (HER2) for disease progression after endocrine therapy. Adult patients with negative advanced or metastatic breast cancer; and Novartis' Kisqali (Ribociclib, ribociclib) in combination with an aromatase inhibitor as first-line therapy for HR-positive and HER2-negative postmenopausal advanced metastatic breast cancer cancer in women; the third is Pfizer’s Ibrance (Palbociclib, Palbociclib), which is used in combination with letrozole for estrogen receptor (ER)-positive, human epidermal growth factor receptor 2 (HER2)-negative Metastatic breast cancer in postmenopausal women.

FGF/FGFR inhibitors suitable for use in the methods of the invention

In some embodiments of the present invention for the treatment of tumors, the other anticancer agent, ie, the anticancer agent used in combination with the anti-PD-1 antibody in addition to the anti-PD-1 antibody, or the anticancer agent used alone is an FGF/FGFR inhibitor. There is no FGF/FGFR inhibitor on the market yet. Boehringer Ingelham's VEGFR/PDGFR/FGFR inhibitor nintedanib for the treatment of liver cancer, non-small cell lung cancer, and idiopathic fibrosis was granted breakthrough drug qualification by the FDA in 2014. In China, dalitinib is currently in the clinical stage.

Uses, Therapies, Drugs and Kits

In one aspect of the present invention, there is provided a method for treating cancer in an individual, the method comprising administering to the individual an anti-PD-1 antibody or antigen-binding fragment thereof and one of a CDK4/6 inhibitor, a FGF/FGFR inhibitor one or more combination therapies.

Combination therapy may also contain one or more additional therapeutic agents. The additional therapeutic agent may be a chemotherapeutic agent or a biotherapeutic agent other than a CDK4/6 inhibitor, FGF/FGFR inhibitor.

In accordance with standard medical practice, each therapeutic agent in the combination therapy of the present invention can be administered alone or in a pharmaceutical composition comprising the therapeutic agent and one or more pharmaceutically acceptable carriers, excipients agent and diluent.

Each therapeutic agent in the combination therapy of the invention can be administered simultaneously, concurrently, or sequentially in any order. The therapeutic agents in combination therapy are administered in different dosage forms, such as one drug is a tablet or capsule and the other is a sterile liquid, and/or are administered at different dosing times, such as chemotherapeutic agents are administered at least daily and The biotherapeutic is administered infrequently, such as once every week, or every two or three weeks.

In some embodiments, the CDK4/6 inhibitor, FGF/FGFR inhibitor is administered prior to administration of the anti-PD-1 antibody, while in other embodiments, the CDK4/6 inhibitor, FGF/FGFR inhibitor is administered Anti-PD-1 antibody was administered afterwards.

In some embodiments, at least one of the therapeutic agents in the combination therapy is administered using the same dosing regimen (dose, frequency, duration of treatment) when the drug is used as a monotherapy to treat the same cancer.

Each small molecule therapeutic agent in the combination therapy described herein can be administered orally or parenterally (eg, intravenous, intramuscular, intraperitoneal, subcutaneous, rectal, topical or transdermal routes of administration).

The combination therapy described herein can be administered before or after surgery, and can be administered before, during, or after radiation therapy.

In some embodiments, the combination therapy described herein is administered to a patient who has not been previously treated with a biotherapeutic or chemotherapeutic agent. In other embodiments, the combination therapy is administered to patients who fail to achieve a sustained response following treatment with a biotherapeutic or chemotherapeutic agent.

The combination therapy of the present invention can be used to treat tumors detected by palpation or by imaging techniques known in the art, such as MRI, ultrasound or CAT scan.

The choice of dosing regimen for the combination therapy of the invention depends on several factors including, but not limited to, serum or tissue conversion rates, degree of symptoms, immunogenicity, and accessibility of target cells, tissues, and organs in the individual being treated. Preferably, the dosing regimen is designed to deliver the maximum amount of each therapeutic agent to the patient with an acceptable degree of side effects. Thus, the dosage and frequency of administration of each biotherapeutic and chemotherapeutic agent in the combination therapy depends on the particular therapeutic agent, the severity of the cancer being treated, and the characteristics of the patient.

One or more of the anti-PD-1 antibody or its antigen-binding fragment and CDK4/6 inhibitor and FGF/FGFR inhibitor according to the present invention can be used as a kit comprising a first container, a second container and a package insert supply.

The first container contains at least one dose of a drug containing an anti-PD-1 antibody or an antigen-binding fragment thereof, and the second container contains at least one dose of a drug containing one or more of CDK4/6 inhibitors, FGF/FGFR inhibitors Drugs, and the package insert or label contains instructions for using the drug to treat cancer. The kit may further comprise other materials useful for administering the drug, such as diluents, filter paper, IV bags and threads, needles and syringes.

Prediction method of anti-PD-1 antibody for cancer treatment effect

A method of predicting the effect of an anti-PD-1 antibody or antigen-binding fragment thereof, particularly toripalimab, of the present invention, administered alone or in combination, on treating cancer in an individual, comprising sequencing the individual prior to treatment. In some embodiments, the cancer is esophageal cancer; in other preferred embodiments, the cancer is ESCC.

In one embodiment, the prediction method of the present invention is a genetic test of an individual before treatment to evaluate whether there is amplification of the gene in the chromosome 11q13 region. In one embodiment, the gene in the chromosome 11q13 region of the individual is not amplified. In another embodiment, the individual has an amplification of the gene in the 11q13 region of chromosome. In one embodiment, the individual has amplification of the CCND1 gene and/or the FGF/FGFR gene. In one embodiment, the individual has CDK4/6 gene and/or FGF3/4/19 gene amplification.

The present invention also includes methods for predicting the therapeutic effect of anti-PD-1 antibodies or antigen-binding fragments thereof administered alone or in combination in tumor patients using genes in the 11q13 region of chromosomes. The absence of gene amplification in the chromosome 11q13 region indicates that the tumor patient is amenable to treatment with anti-PD-1 antibodies or antigen-binding fragments thereof administered alone or in combination.

In certain embodiments, the present invention also provides the use of a detection reagent for a biomarker in the preparation of a kit for predicting the effect of anti-PD-1 antibody on cancer treatment. Such reagents include, for example, reagents that detect the presence or absence of gene amplification of the chromosome 11q13 region in an individual's genes. Preferably, such reagents include, for example, reagents that detect the presence or absence of CCND1 gene and/or FGF/FGFR gene amplification in an individual's genes. More preferably, such reagents include, for example, reagents that detect the presence or absence of CDK4/6 gene and/or FGF3/4/19 gene amplification in an individual's genes.

The present invention also includes the use of the reagent for detecting the gene in the chromosome 11q13 region in the preparation of a kit for predicting the effect of anti-PD-1 antibody in treating cancer. Such reagents include but are not limited to reagents routinely used in tests, including but not limited to primers, probes, reagents required for PCR, and the like.

abbreviation

Throughout the description and examples of the present invention, the following abbreviations are used:

One dose of BID, 2 times a day

CDR complementarity determining regions

FR frame area

IgG Immunoglobulin G

IHC immunohistochemistry

WES whole exome sequencing

PBMC peripheral blood mononuclear cells

OR overall response

ORR objective response rate

OS overall survival

PD disease progression

PFS progression-free survival

DFS disease free survival

DCR disease control rate

PR partial response

CR complete response

SD stable disease

DLT dose-limiting toxicity

MTD maximum tolerated dose

AE Adverse Events

Q2W One dose every two weeks

QD one dose per day

TRAE Treatment-Related Adverse Reactions

SAE Serious Adverse Reactions

TMB tumor mutational burden

EC Esophageal Cancer

ESCC esophageal squamous cell carcinoma

LDH lactate dehydrogenase

ECOG Eastern Cooperative Oncology Group

The present invention is further illustrated by the following examples, which should not be construed as limiting the invention. The contents of all references cited throughout this application are expressly incorporated herein by reference.

Example

Example 1: Clinical study of anti-PD-1 antibody alone in the treatment of tumors

Inclusion criteria: Eligible subjects must be (1) aged between 18 and 75 years old, (2) histologically and/or cytologically confirmed to have advanced and/or metastatic ESCC, (3) patients with advanced ESCC , must have received or at least one treatment has progressed (including but not limited to chemotherapy or radiotherapy), (4) ECOG score of 0 or 1.

Subjects must have evaluable lesions according to RECIST v 1.1 criteria, no other previous or concurrent malignancies, no active autoimmune disease or history of autoimmune diseases, and no need for long-term immunosuppression The concomitant diseases of drug treatment are not allowed to receive anti-CTLA4, anti-PD-1, anti-PD-L1 antibody treatment in the past, not allowed to have active hepatitis B or hepatitis C virus infection, not allowed to be pregnant or breastfeeding, not allowed to be enrolled in the group Antineoplastic therapy, radiotherapy or any surgical treatment in the previous 4 weeks. The demographics of the enrolled subjects are shown in Table 1.

Table 1: Demographics of enrolled subjects

Notes: *Positivity is defined as ≥1% tumor cells or immune cells stained with SP142 IHC; Abbreviations: ECOG, Eastern Cooperative Oncology Group; LDH, Lactate Dehydrogenase.

Tested drug: anti-PD-1 antibody Toripalimab (Toripalimab) (WO2014206107).

The anti-PD-1 antibody dose group in this experiment is: this study firstly conducts the 3 mg/kg dose group study. After enrollment, subjects will receive treatment every 2 weeks (Q2W), every 4 weeks as a cycle, until disease progression, unacceptable toxicity occurs, subject withdraws consent, and the investigator judges that there is no further benefit , or death.

Clinical Design:

This is an open-label, multicenter clinical phase Ib/II trial divided into 8 independent cohorts, and the content of the present invention mainly evaluates the results of cohort 2. To evaluate the safety, tolerability, and antitumor activity of anti-PD-1 antibodies in the treatment of chemotherapy-refractory advanced ESCC.

From 2017.3.9 to 2017.8.24, a total of 60 subjects with advanced ESCC were enrolled, of which 59 subjects were treated with toripalimab and 1 subject withdrew their consent before treatment.

The planned dose is 3 mg/kg, Q2W.

1.1 Safety Research:

As of December 31, 2018, the last patient was enrolled for 16 months, and 59 patients received at least one dose of toripalimab and were included in the safety analysis. Treatment-related adverse events (TRAEs) occurred in 56 of 59 patients (94.9%), but most TRAEs were grade 1 or 2 (Table 2). Common TRAEs included weight loss (18.6%), anemia (18.6%), decreased appetite (18.6%), fever (16.9%), cough (16.9%), leukopenia (15.3%), increased AST (13.6%) , hypothyroidism (13.6%), increased ALT (11.9%), fatigue (11.9%), nausea (11.9%), increased total bilirubin (10.2%), low hemoglobin count (10.2%) and constipation ( 10.2%) (Table 2). Grade 3 and higher TRAEs occurred in 19 (32.2%) patients. Eleven (18.6%) patients had grade 3 TRAEs, including 2 anemia, 2 hyponatremia, 1 hypertension, 1 anorexia, 1 dysphagia, 1 esophageal stricture, 1 fatigue, 1 Cases of hydropericardium, 1 case of hypercalcemia, 1 case of leukopenia, 1 case of upper respiratory tract infection and 1 case of neck infection. Grade 4 TRAEs occurred in 2 (3.4%) patients, including 1 with hyperuricemia and 1 with high paraplegia. All six treatment-related deaths were potentially unrelated to treatment, including one case of cachexia, two cases of pneumonia, and three cases of unknown etiology. 14 (23.7%) patients permanently discontinued due to TRAE; 6 (10.2%) patients experienced dose interruption due to TRAE. It can be seen that toripalimab has a manageable safety profile in ESCC patients.

Table 2: Common Treatment-Related Adverse Reactions in the Cohort (>10%)

1.2 Antitumor activity studies:

By 2018.12.31, 67.8% (40/59) of the subjects had died. Follow-up was discontinued in 5.1% (3/59), consent was withdrawn in 11.9% (7/59), treatment was discontinued in 74.5% (44/59), and 8.5% (5/59) were still on study.

The median duration of treatment was 3.5 months (range 0.1-19.1 months).

As of the data cutoff date, investigators assessed using RECIST v1.1, with a total of 1 CR, 10 PRs (including 2 undiagnosed PRs), and 17 SDs among 59 patients. The best response rate was 18.6% (95% CI 9.7 to 30.9) with a DCR of 47.5% (95% CI 34.3 to 60.9). Patients with CR had previously received 1-line chemotherapy and no radiotherapy. Of the 10 PR patients who had received prior radiotherapy, 4 had also received 2 prior systemic chemotherapy, and the other 6 had received 3 or 4 prior chemotherapy.

Progression-free survival and overall survival

The confirmed ORR was 15.3% (95% CI 7.2 to 27.0). The average response time was 1.8 months. The median duration of response was 11.2 months. Any reduction in target lesion size relative to baseline was observed in 25 (42.4%) subjects (Figures 1A and 1B). The median PFS was 2.1 months and the median OS was 6.9 months (Figures 1C and 1D).

Example 2: Corresponding relationship between PD-L1 expression and anti-PD-1 antibody in the treatment of tumors

Archived or fresh tumor biopsies were performed on subjects prior to toripalimab treatment in the clinical trial described in Example 1. Detection of PD-L1 in formalin-fixed paraffin-embedded (FFPE) tumor tissue samples by immunohistochemistry (IHC) and validated on the VENTANA Benchmark Ultra system at the Clinical Central Laboratory (Q2 Laboratory, Beijing) . PD-L1 immunohistochemical detection was performed using Spring Bioscience (Roche) rabbit anti-human PD-L1 monoclonal antibody (clone SP142, Cat No: M4422). Expression of PD-L1 on tumor cells (TC) and tumor-infiltrating immune cells (IC) was assessed by certified pathologists using stained tumor tissue. PD-L1 positive status was defined as the presence of ≥1% of tumor cells with membrane staining intensity or the presence of PD-L1 staining intensity of tumor-infiltrating immune cells covering ≥1% of occupied, intratumorally associated, continuous pericancerous stromal tumors area.

PD-L1 expression assays were performed on tumor biopsy samples taken from the 57 subjects in Example 1. Nineteen (33.3%) PD-L1 positive and 38 (66.7%) PD-L1 negative samples were identified (Fig. 2A).

PD-L1 positivity was defined as ≥1% tumor cell (TC) or immune cell (IC) positivity using SP142IHC staining.

In this study, there were no significant differences in ORR (15.8% vs. 18.4%) or OS (6.7 months vs. 6.9 months) between PD-L1-positive patients and PD-L1-negative subjects (Figures 2A, 2C, 2D). ). The median PFS of PD-L1-positive subjects was numerically better than that of PD-L1-negative subjects (3.4 months vs. 2.0 months), but the difference was not statistically significant (p=0.85) (Fig. 2C).

Of the tumor biopsies in this study, only 5 (8.8%) subjects had more than 5% positive PD-L1 expression (Figure 2A). Subjects with >5% PD-L1 expression had better clinical responses than those with low or no PD-L1 expression, with 40% ORR in PD-L1 positive subjects and 15.4% in 100% DCR versus negative subjects % ORR and 42.3% DCR. The difference in ORR was not statistically significant (p=0.21), and the difference in DCR was statistically significant (p=0.019).

Example 3: Corresponding relationship between tumor mutational burden (TMB) and anti-PD-1 antibody in tumor therapy

During the clinical trial described in Example 1, whole exome sequencing (WES) was performed on tumor biopsy samples taken from 50 subjects. TMB is determined by analyzing somatic mutations within coding regions of the human genome. Valid TMB results were obtained in 47 patients (Table 3).

In this study, TMB was generally lower in ESCC patients. None of the biopsy samples had more than 20 mutations per megabase pair, and only 13 biopsy samples had 10-20 mutations per megabase pair.

Using 12 mutations per Mb as a cutoff, patients with TMB greater than or equal to 12 mutations/Mb (n=11) had similar ORRs (18.2% ratio) to those with TMB less than 12 mutations/Mb (n=36). 19.4%) (Figure 2B). The group with high TMB (≥12) had numerically better PFS (4.0 months vs. 1.9 months) and OS (11.5 months vs. 6.7 months) than the group with low TMB (<12), although this The differences were not statistically significant (Figures 2E and 2F).

Table 3: Corresponding relationship between TMB and anti-PD-1 antibody in the treatment of tumors

Example 4: Panorama of mutations in anti-PD-1 therapy and its corresponding relationship with clinical efficacy

4.1 Whole exome sequencing (WES) and data analysis

Tumor tissue and matched PBMC samples were collected, and 4 μm sections of hematoxylin and eosin-stained FFPE samples were examined by a pathologist to ensure that each had greater than 80% nucleated cellularity and no tumor content. less than 20%. Use enough unstained FFPE sections to extract no less than 200 ng of DNA. The reference was matched from approximately 200 μl of PBMC matched to typically generate 1-5 μg of DNA.

Library preparation uses sonication to disrupt double-stranded DNA to 250 bp. Libraries for end repair, dA addition, and adapter ligation were subsequently performed using the SureSelect XT library prep kit, or the KAPA Hyper prep kit (KAPA Biosystems), followed by PCR amplification and quantification by Qubit assessment. Follow the protocol to hybridize the capture region of interest and prepare the library. Sequence reads were generated using an Illumina NovaSeq 6000 (Illumina Inc., San Diego, CA). Check and control data quality and build a custom bioinformatics pipeline for SNVs, long and short indels, CNAs, gene rearrangements, TMBs and MSIs. Finally, all detected mutations were clinically treated against our internal annotated database. Based on the annotated mutations, a concise report based on clinical trials and literature will be generated.

4.2 Identification of somatic signal-to-noise ratios, short indents and copy number alterations (SCNAs)

The original reads were aligned with the human genome reference sequence (hg19) using a Burrows-Wheeler sequencer, and then PCR repeats were removed using the Picard tag replication algorithm. The original calls of SNVs and S-indels are called by internal algorithms. Supporting optional calls requires at least 5 reads. Variants with read depths less than 50x, strand bias less than or greater than 90%, and VAF < 1% were removed. General SNPs defined as frequencies exceeding 1.5% from the dbSNP database (version 147) or the Exome Sequencing Project 6500 (ESP6500) or frequencies exceeding 1.5% from the 1000 Genomes Project were also excluded for further consideration. Products from sequencing errors or populations were also excluded.

To define SCNA events, aligned reads within each exonic region were normalized, and the results were further corrected for GC content and reference genome mappability using CNVKIT (version 0.9.1). The obtained duplication rates are segmented using a cyclic binary segmentation algorithm. The proportion of aberrant tumor cells was estimated using the allele frequencies of over 4000 sequenced SNPs (single nucleotide polymorphisms) following the ASCAT method. After depth normalization, GC, mappability correction, the copy rate of tumor tissue and matched normal blood samples was calculated. Fragments were considered amplified or deleted if log2 (copy rate) exceeded 0.8 or did not exceed -1, respectively.

The genome map of WES revealed that missense mutations or truncations in TP53 (p53) (76%), RYR2 (22%), NOTCH1 (20%), LRP1B (17%) and TRIO (17%) were ESCC in this study The top five mutated genes in tumor biopsies (Figure 3). But missense mutations or truncations or other loss-of-function mutations in these five genes were not associated with clinical response.

Example 5: Relationship between 11q13 gene amplification and resistance to anti-PD-1 therapy

rRNA消耗试剂盒(cat#E6310L,新英格兰生物实验室)进一步处理以删除rRNA。分别使用M-MLV RT RNase(H-)(cat#M3683,Promega公司)和NEB第二链mRNA合成试剂盒(cat#E6111L,新英格兰生物实验室)合成第一链和第二链cDNA。对cDNA产物进行超声处理产生～200bp的片段(CovarisE220)。适配器连接的库是由cDNA使用KAPA Hyper制备试剂盒(cat#07962363001,罗氏)创建，在NovaSeq 5000/6000平台上测序。每个注释转录本的相对丰度以百万分之转录量(TPM)和分析前的log2转化表示。Whole transcriptome sequencing (RNA-seq) was performed using the following method. RNA was extracted from unstained FFPE fractions using the miRNeasy kit (cat#217504, Qiagen), and then used

The rRNA depletion kit (cat#E6310L, New England Biolabs) was further processed to delete rRNA. First-strand and second-strand cDNA were synthesized using M-MLV RT RNase (H-) (cat#M3683, Promega Corporation) and NEB Second-Strand mRNA Synthesis Kit (cat#E6111L, New England Biolabs), respectively. Sonication of the cDNA product yielded a ~200 bp fragment (CovarisE220). Adapter-ligated libraries were created from cDNA using the KAPA Hyper prep kit (cat#07962363001, Roche) and sequenced on the NovaSeq 5000/6000 platform. The relative abundance of each annotated transcript is expressed in transcripts per million (TPM) and log2 transformation before analysis.

The WES analysis of Example 4 showed that 24 of the 50 biopsies (48.0%) contained an amplification of the chromosome 11q13 region (FIG. 4A). Messenger RNA expression analysis further confirmed the genomic DNA amplification event, and mRNA levels correlated with the amplification status of corresponding genes in this region, including CCND1 (Cyclin D1) and FGF family members (FGF3/4/19) (Fig. 4B). Importantly, patients without 11q13 amplification (n=26) had a statistically significantly better objective response rate than patients with 11q13 amplification (n=24) (30.8% vs 4.2%, p=0.024) ( Figure 5A) and longer PFS (3.7 months vs 1.0 months; HR=0.47 [95% CI 0.24 to 0.91], p=0.025). Patients without 11q13 amplification also had longer median OS (11.5 months vs. 5.6 months; HR=0.60 [95% CI 0.30 to 1.20]) (Figure 5C).

Example 6: Additional Biomarkers and Subgroup Analysis

Additional biomarkers or subgroup analyses associated with clinical outcomes included: age, sex, ECOG score, prior treatment, presence of liver metastases at baseline, tumor volume, and baseline serum LDH levels. Of these, patients with an ECOG score of 0, low baseline tumor volume, and baseline LDH levels above the upper limit of normal had numerically better ORR. However, these differences were not statistically significant (Table 4).

Table 4: Correlation of other markers and subgroups with clinical efficacy

Claims (13)

1. Use of an anti-PD-1 antibody and/or an antigen-binding fragment thereof alone, or an anti-PD-1 antibody and/or an antigen-binding fragment thereof in combination with an anti-cancer agent other than an anti-PD-1 antibody, in the manufacture of a medicament for treating a solid tumor that does not have gene amplification of the region of chromosome 11q 13.

2. The use of claim 1, wherein the anti-cancer agent is a small molecule targeted anti-cancer agent.

3. The use of claim 1, wherein the anti-cancer agent is selected from one or more of a CDK4/6 inhibitor, FGF/FGFR inhibitor.

Use of one or more of a CDK4/6 inhibitor, FGF/FGFR inhibitor, alone or in combination with an anti-PD-1 antibody and/or antigen-binding fragment thereof, in the preparation of a medicament for the treatment of a solid tumor having a gene amplification of the chromosome 11q13 region.

5. The use of any one of claims 1 to 4, wherein the anti-PD-1 antibody is a monoclonal antibody or an antigen-binding fragment thereof that specifically binds PD-1 and blocks the binding of PD-L1 to PD-1.

6. The use of claim 5, wherein the light chain complementarity determining region of the anti-PD-1 antibody is as set forth in SEQ ID NO: 1.2 and 3, and the heavy chain complementarity determining region is shown in SEQ ID NO: 4.5 and 6.

7. The use of claim 5, wherein the light chain variable region of the anti-PD-1 antibody is as set forth in SEQ ID NO: 7, the heavy chain variable region is shown as SEQ ID NO: 8 is shown in the specification; or the light chain of the anti-PD-1 antibody comprises the amino acid sequence shown in SEQ ID NO 9, and the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 10, or a pharmaceutically acceptable salt thereof.

8. The use of claim 5, wherein the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, terliplizumab, certralizumab, carprilizumab and tirlizumab, or a combination thereof.

9. The use according to claims 1-4, wherein the solid tumor is esophageal cancer.

10. The use according to claim 9, wherein the solid tumor is esophageal squamous cell carcinoma.

11. Use of a reagent for detecting gene amplification in chromosome 11q13 region in the preparation of a test kit for predicting the effect of a tumor patient on treatment with an anti-PD-1 antibody and/or an antigen-binding fragment thereof.

12. Use of a reagent for detecting CDK4/6 gene amplification in the preparation of a test kit for predicting the effect of a treatment of a tumor patient with an anti-PD-1 antibody and/or an antigen-binding fragment thereof.

13. Use of a reagent for detecting FGF/FGFR gene amplification in the preparation of a test kit for predicting the effect of a tumor patient on treatment with an anti-PD-1 antibody and/or an antigen-binding fragment thereof.

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